Analysis of adaptive signaling in response to targeted therapies can provide insight into mechanisms of resistance and appropriate combinations of therapeutic agents. In a screen for adaptive resistance mechanisms in BRAF mutant melanomas, we made a serendipitous discovery that may provide a path to targeted therapy for melanomas driven by mutant NRAS, a disease for which there are few options. We found that a combination of a RAF inhibitor (PLX4720) and a HER-family inhibitor (lapatinib) could kill NRAS mutant melanomas that were resistant to either drug alone. The mechanism of killing did not require inhibition of the MAP Kinase or the PI3 Kinase pathways. The data strongly suggest that either RAF or a HER family receptor (or both) are engaged in non-canonical signaling activity that provides a previously undetected cytotoxic vulnerability. This proposal aims to identify the molecular basis for that non-canonical signaling, and determine the most appropriate setting for in vivo studies and a clinical trial. Aim 1. We will analyze the signaling network that links BRAF, NRAS and HER family members to identify signaling nodes critical for the PLX4720-lapatinib synergy. We will utilize proteomic techniques (Reverse Phase Protein Micro Arrays; RPPA) and gene expression arrays to determine the adaptive responses to drug treatments made in the phosphoproteome and the transcriptome. We also will analyze the mode(s) of cytotoxicity induced by combined therapy (apoptosis, cell cycle arrest, autophagy). These responses will be analyzed using publicly available computational tools as well as integrated pathway analysis tools developed by our collaborators at the University of Virginia. We will explicitly examine our -omic analyses for hypothesized mechanisms including functional interactions of CRAF or BRAF with mitochondria, Protein Kinase A and the spindle assembly checkpoint; and of HER family kinases with the HIPPO pathway, RAC, PAK, Reactive Oxygen, Calcium, and ERK5. Aim 2. We will test the functional significance of signaling nodes identified in Aim 1. We will perform epistasis experiments, complementing cytotoxicity with RNAi and rescuing inhibited growth with ORFeome expression. We also will determine whether appropriate small molecules can phenocopy the cytotoxic effects of the PLX4720-lapatinib combination. Adaptive homeostatic responses are a major confounder for targeted therapies, and the information obtained here will provide a strong foundation for therapeutic application with FDA-approved drugs and validated targets.